Mosquito-borne viral diseases remain significant causes of morbidity and mortality throughout much of the world and impose tremendous burdens on human and animal care infrastructures. It is critical that we have a better understanding of the molecular basis of vector competence and arbovirus transmission by mosquitoes. Genome sequence information and other bioinformatics are available for three major vector species (Anopheles gambiae, Aedes aegypti and Culex pipiens). New techniques are still needed by vector biologists and arbovirologists to fully exploit this explosion of information and develop a more complete understanding of mosquito-borne disease transmission. For the last 15 years, AIDL researchers have been at the forefront developing molecular tools termed alphavirus transducing systems or ATS's that allow gene expression in a variety of mosquito (and insect) species. ATS's have facilitated gene expression in adult mosquitoes, allowed functional analyses of vector and alphavirus genes, and have been used in the study of mosquito-alphavirus interactions. Vector biologists at AIDL, and elsewhere, are currently manipulating innate immune pathways in mosquitoes and need tools such as ATS's to characterize the effects of these manipulations on arbovirus transmission. Virologists here and at other institutions are testing new antiviral vaccines and drugs in animal models and need tools that facilitate virus exposure of animals by the natural route of infection. We think that ATS's technology coupled with fluorescent and bioluminescent technology can be an important addition in our arsenal of tools that allow us to observe (in real time) arbovirus transmission. This approach will have an added benefit of reducing the numbers of animals that are needed for transmission and pathogenesis analyses and in evaluating antiviral protection in animals. The specific aims of this grant renewal are as follows: 1) use ATS's to develop mosquito transmission models for alphaviruses, 2) use these transmission models to understand and improve ATS's induction of RNA interference (RNAi) in mosquitoes and its impact on transmission, and 3) generate novel mosquito reporters of alphavirus infection and transmission. PUBLIC HEALTH RELEVANCE Mosquito-borne viral diseases are significant causes of illness and death throughout much of the world. We are only now beginning to understand how virus and mosquito genetics influence vector infection and virus transmission to a host. In this proposal we will develop new alphavirus-based tools to help us understand the effects of these virus and mosquito genetic factors on infection and transmission. We will develop new methods to track virus transmission from mosquitoes to experimental animals in such a way that the entire transmission cycle may be directly visualized. In this study, we plan to use fluorescent and bioluminescent technology to readily observe and detect alphavirus transmission from mosquitoes to small animals. This grant proposal will use alphavirus transducing systems (ATS's) to develop vector transmission models, identify specific alphavirus/mosquito interactions affecting virus transmission, improve ATS's ability to knock down targeted genes, and develop novel ATS-based biomarkers for mosquito infection.